Methods of genetic engineering

Methods of genetic engineering

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Induction and repression

The bacterial metabolism often has to be adapted to a rapidly changing environment, for example when a new usable substrate is available or a previously used feed source dries up. This requires a precise adaptation of protein biosynthesis, which is mainly regulated in a coordinated manner at the level of transcription, i.e. via the induction or repression of gene expression.

Regulatory proteins act as mediators between the environment and protein biosynthesis. They bind to DNA regions in the vicinity of a gene or operon and influence the binding properties of RNA polymerase. The promoter is a base sequence that is recognized and bound by RNA polymerase; this is where the transcription begins. The operator is a base sequence which lies between the promoter and the structural genes and which can bind a repressor. When a repressor binds to the operator region, transcription by the RNA polymerase is prevented. An inductor can in turn change the conformation of the repressor so that it no longer binds and leaves the operator's area. The regulation via induction and repression can be illustrated well using the example of the lac- demonstrate operons.

That lac-Operon is for the breakdown of the sugar lactose in lactose E. coli responsible. The disaccharide lactose is taken up by the permease (LacY) and cleaved by the β-galactosidase (LacZ), while the transacetylase (LacA) is involved in the breakdown of related sugar compounds. If there is no lactose around the bacterium, it willlac-Operon repressed by the repressor LacI. In the presence of lactose the lac-Operon derepresses unless glucose is available at the same time. In this case, the glucose is first broken down before the proteins for the lactose breakdown are synthesized.

P = promoter, O = operator, black: termination signal. Structural genes: lacZ (β-galactosidase), lacY (permease), lacA (transacetylase) and lacI (Lac repressor).

Application of the lac-Operons in molecular biology

Components of the lac-Operon-based systems have been used for many years in molecular biology for various questions: For the expression of proteins, expression vectors are often used whose control elements are derived from thelac-Operon originate.

That lacZ-gene itself is often used as a reporter gene to test the activity of a promoter. In addition to the natural lactose substrate, β-galactosidase also cleaves colored substrates such as X-gal (5-bromo-4-chloro-3-indolyl-β-galactopyranoside; the product is colored blue) or ONPG (ortho-Nitrophenyl-galactoside). The colorless ONPG is enzymatically transformed into the intense yellow one ortho-Nitrophenol converted, the concentration of which can be determined photometrically. The amount of dye formed is dependent on the β-galactosidase concentration and, in comparison with a standard, allows conclusions to be drawn about the activity of an unknown promoter.

Video: Methods of Functional Annotation of Genetic Variants Webinar (July 2022).


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